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V J Hruby

Publications and source records attributed to V J Hruby.

At least 145 records · Page 8Linked to original sources
PubMed1995

Properties of TAN-67, a nonpeptidic delta-opioid receptor agonist, at cloned human delta- and mu-opioid receptors.

2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino[2,3,30g]isoquinoline (TAN-67) is a nonpeptidic delta-opioid receptor agonist. This report describes its receptor binding affinity and agonist potency at human and mouse delta and mu-opioid receptors. The binding affinities of TAN-67 and the cyclic enkephalin analog, (D-Pen2, 4'-Cl-Phe4, D-Pen5]enkephalin (pCl-DPDPE) were measured by radioligand binding inhibition studies at mouse and human variants of the delta and mu-opioid receptor using [3H]Naltrindole and [3H]D-Phe-Cys-Tyr-D-Trp-Orn-Thr -Pen-Thr-NH2, respectively. TAN-67 showed high binding affinity (Ki = 0.647 nM) at the human delta-opioid receptor and high delta-opioid receptor binding selectivity ( > 1000-fold) relative to the human mu-opioid receptor. TAN-67 also showed high potency (EC50 = 1.72 nM) for the inhibition of forskolin-stimulated cAMP accumulation at human delta-opioid receptors expressed by intact Chinese hamster ovary cells but low potency (EC50 = 1520 nM) at human mu-opioid receptors expressed by intact B82 mouse fibroblast cells. The results show that TAN-67 has similar binding affinities, selectivity and potencies as pCl-DPDPE at human delta and mu-opioid receptors. These results combined with the nonpeptidic structure of TAN-67 suggest that this compound has therapeutic potential as a delta-opioid receptor agonist.

Analgesics↗
PubMed1995

Cyclic lactam alpha-melanotropin analogues of Ac-Nle4-cyclo[Asp5, D-Phe7,Lys10] alpha-melanocyte-stimulating hormone-(4-10)-NH2 with bulky aromatic amino acids at position 7 show high antagonist potency and selectivity at specific melanocortin receptors.

The cloning of the melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) receptors (MC1-R and MC2-R, respectively) recently has led to the identification of three additional melanocortin receptors, MC3-R, MC4-R, and MC5-R. The MC2 receptor primarily recognizes only ACTH peptides, but the other four receptors all recognize alpha-melanocyte-stimulating hormone (alpha-MSH) and potent alpha-MSH agonists such as [Nle4,D-Phe7]alpha-MSH-NH2 and Ac-Nle4-c[Asp5,D-Phe7,Lys10]alpha-MSH-(4-10)-NH2 as well as ACTH. The absence of any known physiological role for these new receptors, expressed both in the brain (MC3-R and MC4-R) and throughout a number of peripheral tissues (MC5-R), has necessitated as search for potent and receptor selective agonists and antagonists. We report here that analogues of the superpotent cyclic agonist analogue Ac-Nle4-c[Asp5,D-Phe7, Lys10]alpha-MSH-(4-10)-NH2, in which a bulky aromatic amino acid is substituted in the 7-position, can produce potent and selective antagonists for melanocortin receptors. Thus, the D-p-iodophenylalanine7-containing analogue Ac-Nle4-c[Asp5,D-Phe(pI)7,Lys10]alpha-MSH-(4-10)-NH2 is a potent antagonist (pA2 = 10.3) in the classical frog skin (Rana pipiens) assay (MC1-R), as is the D-2'-naphthylalanine7 (D-Nal(2)7)-containing analogue Ac-Nle4-c[Asp5,D-Nal(2)7,Lys10]alpha-MSH-(4-10)-NH2 (pA2 > 10.3). Interestingly, the D-p-chloro- and D-p-fluorophenylalanine7-containing analogues lacked antagonist activities at all melanotropin receptors, and both exhibited full agonist potency in the frog skin assay. The activity of these analogues also was examined at four mammalian melanocortin receptors. Interestingly, Ac-Nle4-c[Asp5,(D-Nal(2)7,Lys10] alpha-MSH-(4-10)-NH2 was found to be a potent antagonist of the MC4-R (pA2 = 9.3) with minimal agonist activity, a less potent antagonist of the MC3-R (pA2 = 8.3) with minimal agonist activity, and a full agonist of the MC1 and MC5 receptors. Surprisingly, Nle4-c[Asp5,D-Phe(pI)7,Lys10]alpha-MSH was found to be a potent agonist at the cloned human MC1-R (EC50 = 0.055 nM) and mouse MC1-R (EC50 = 0.19 nM) but had potent antagonist activities at the human MC4-R (pA2 = 9.7) and human MC3-R (pA2 = 8.3) with significant partial agonist activities (EC50 = 0.57 and 0.68 nM, respectively) as well. Thus, highly potent and receptor selective antagonist analogues can arise from substitution of the D-Phe7 residue with a bulky aromatic amino acid. These analogues can be used to help determine the functional roles of these receptors.

Amino Acid Sequence↗
PubMed1995

Synthesis using a Fmoc-based strategy and biological activities of some reduced peptide bond pseudopeptide analogues of dynorphin A1.

Eight analogues of Dyn A(1-11)-NH2 incorporating the enzymatically stable psi(CH2-NH) isosteric peptide bond replacement were synthesized and tested for binding affinity at the central opioid mu, delta, and kappa receptors in guinea pig brain (GPB) homogenates and for activity at the peripheral kappa (and mu) receptors in the guinea pig ileum (GPI). The peptidic analogues were synthesized by solid phase techniques using a Fmoc/tert-butyl strategy, and the psi(CH2-NH) bond, or reduced bond, was introduced via reductive alkylation of the N-terminal amino group of the growing peptide with a Fmoc-N(alpha)-protected amino aldehyde. The synthesis of Fmoc-N(alpha)-protected amino aldehydes also is described. Several other peptides have been previously synthesized incorporating this modification and showed for instance increased enzymatic stability and antagonist properties. Results obtained in the GPB show that modifications of the peptide bond in the address site (analogues 4-9) do not affect the binding at the kappa receptor and, with a few exceptions, at the mu and delta receptors. On the other hand, analogues 2 and 3, modified in the message segment of Dyn A(1-11)-NH2, show a decrease in binding affinity at all three receptors. In the GPI, the results are more varied as the influence of the peptide bond modification seems to be more important than in the GPB. Finally, selected analogues were tested with no indication for antagonist activity at the kappa peripheral receptor.

Amino Acid Sequence↗
PubMed1995

Design, synthesis, biology, and conformations of bicyclic alpha-melanotropin analogues.

Seven side chain-constrained bicyclic alpha-melanotropin (alpha-MSH) analogues were designed and synthesized, their conformations analyzed, and their biological properties examined in the frog skin and lizard skin bioassays. The structure of these analogues is based on the central sequence Ac-Cys4-Xaa5-His6-DPhe7-Arg8-Trp9-Cys10-Lys11 -NH2 (Xaa5 = Asp or Glu) and has been extended on the N-terminal with the amino acids Ser1-Tyr2-Ser3 and on the C-terminal with Pro12-Val13 to more closely resemble the native hormone alpha-MSH. The analogue Ac-Cys4-Asp5-His6-DPhe7-Arg8-Trp9-Lys10-Cys11 -NH2 also was synthesized, and its conformational and biological properties were examined. Design of these analogues was based upon the previously identified superpotent monocyclic peptides [Cys4,DPhe7,Cys10]alpha-MSH(4-10)-NH2 and [Nle4,Asp5,DPhe7,Lys10]alpha-MSH(4-10)-NH2 with the rationale of increasing conformational constraints to restrict the available backbone conformations as a means to identify the conformations that facilitate biological activity. Computer-assisted conformational analysis of the central tetrapeptide residues 6-9 identified beta-turns which varied with respect to the residue in the i + 1 position. Each highly constrained peptide contains D-Phe7 and a 23-membered ring which has previously been identified as crucial to produce prolonged acting peptides with superagonistic activities. The bicyclic peptides reported in this study are full agonists and are 25-400-fold less potent than alpha-MSH in the frog and lizard skin bioassays.

Amino Acid Sequence↗
PubMed1995

The synthesis and opioid receptor binding affinities of analogues of dermorphin and its N-terminal tetrapeptide fragment with dibasic acids in position 2.

Analysis of possible mu opioid receptor active conformations for dermorphin suggested that the topographical location of the tyramine moiety of the N-terminal tyrosine can be simulated with the phenol of tyrosine or desamino-tyrosine (4-hydroxyphenylpropionic acid) and a basic group located on the side chain of a dibasic acid residue located in position 2. The biological properties of respective analogs with D- or L-arginine, and D- or L-lysine in the position 2 of dermorphin or desamino-dermorphin and their N-terminal tetrapeptide fragments, has provided evidence in support of this prediction, and questions the dogma that an N-terminal tyrosine is a necessary element for opioid agonist peptides.

Amino Acid Sequence↗
PubMed1995

[125I]SNF 8702: a selective radioligand for CCKB receptors.

The CCK-8 analogue, SNF 8702, was radioiodinated. [125I]SNF 8702 showed high-affinity specific binding for both guinea pig and mouse brain tissues, whereas no specific binding was seen for guinea pig pancreatic tissue. The properties of the site labeled by [125I]SNF 8702 were characterized by binding inhibition studies for a series of CCKA and CCKB receptor ligands. The binding selectivity profile corresponded to that for the CCKB receptor. The labeled compound is stable for more than 6 weeks during storage at -20 degrees C.

Amino Acid Sequence↗
PubMed1995

In vitro stability of some reduced peptide bond pseudopeptide analogues of dynorphin A.

Eight analogues of DYN A(1-11)-NH2 incorporating the nonhydrolyzable psi [CH2-NH] peptide bond surrogate were tested for their in vitro enzymatic stability in mouse brain homogenates. Results show that the Leu(5)-Arg6 and to a lesser extent the Arg(7)-Ile8 and Ile(8)-Arg9 peptide bonds are the more susceptible to enzymatic cleavage in the native peptide. (Leu5 psi[CH(2)-NH]Arg6)DYN A(1-11)-NH2 exhibits an almost complete resistance to enzymatic cleavage with a half-life greater than 500 min in brain, compared to 42 min for the standard peptide, DYN A(1-11)-NH2.

Animals↗
PubMed1995

Molecular biology and pharmacology of cloned opioid receptors.

The cloning and expression of DNA for the three major opioid receptor types (mu, delta, and kappa) present new research opportunities for the characterization of opioid drugs and their interactions with these receptors. Genomic and cDNA clones for opioid receptors exist for several animal species including mouse, rat, guinea pig, and human. These include clones for all three human opioid receptor types. The receptor proteins consist of about 400 amino acids and have the characteristic seven transmembrane domain structure of G-protein-coupled receptors. There is about 60% amino acid identity between opioid receptor types and about 90% identity between a receptor type cloned from different animal species. All opioid receptor types mediate the inhibition of adenylyl cyclase in response to agonist binding. Radioligand binding and functional studies using the cloned receptors tend to support current conclusions on opioid drug receptor selectivity and activity. Investigations of opioid receptor chimeras and single amino acid mutants are providing information on the ligand recognition sites of these receptors and essential support for the development of computational opioid receptor models. A molecular model of the human delta opioid receptor is included in this review.

Amino Acid Sequence↗
PubMed1995

The melanotropic peptide, [Nle4,D-Phe7] alpha-MSH, stimulates human melanoma tyrosinase activity and inhibits cell proliferation.

Seventeen human melanoma cell (HMC) lines, both melanotic and amelanotic, were incubated in the continuous presence of a potent melanotropic peptide hormone analog, [Nle4,D-Phe7] alpha-MSH, for 72 hr with daily changes of medium. Only one cell line (HD, melanotic) consistently responded to the hormone analog by increased tyrosinase activity. Three (one melanotic, two amelanotic) of the HMC lines also failed to respond to the peptide by either increased or decreased enzyme activity when incubated continuously in the presence of the peptide for longer periods of time (6,15,27,43 days). The HD cell line, however, again responded with increasingly enhanced basal enzyme activity the longer the cells were incubated in the presence of the melanotropin. One amelanotic cell line (C8161) responded with enhanced enzyme activity when grown to confluency in the continuous presence of the peptide. Basal tyrosinase activity of the C8161 cell line may have increased as cell density in the flasks increased. These results suggest that under conditions of increased cell number, phenotypic expression of tyrosinase activity in so called "amelanotic" (tyrosinase-negative) cells is increased and can be enhanced further by stimulation with a melanotropic peptide. Under conditions of increased cell number, the presence of [Nle4,D-Phe7] alpha-MSH caused morphological differentiation (shape change); the cells became enlarged and very dendritic. The number of cells in monolayer (surface of the flask) and in the medium were drastically reduced in both melanotic and "amelanotic" cell lines incubated with [Nle4,D-Phe7] alpha-MSH. The data support other published reports that melanotropic peptides inhibit human melanoma cell growth (proliferation) in vitro, most likely through a cytostatic mechanism. [Nle4,D-Phe7] alpha-MSH also exhibited a prolonged (residual) inhibitory action on HD cell proliferation. In other words, inhibition of cell growth (proliferation) of the HMCs was evident even several days after removal of the melanotropic peptide from the incubation medium.

Animals↗
PubMed1995

SNC 80, a selective, nonpeptidic and systemically active opioid delta agonist.

The present study has investigated the pharmacology of SNC 80, a nonpeptidic ligand proposed to be a selective delta agonist in vitro and in vivo. SNC 80 was potent in producing inhibition of electrically induced contractions of mouse vas deferens, but not in inhibiting contractions of the guinea pig isolated ileum (IC50 values of 2.73 nM and 5457 nM, respectively). The delta selective antagonist ICI 174,864 (1 microM) and the mu selective antagonist CTAP (1 microM) produced 236- and 1.9-fold increases, respectively, in the SNC 80 IC50 value in the mouse vas deferens. SNC 80 preferentially competed against sites labeled by [3H]naltrindole (delta receptors) rather than against those labeled by [3H]DAMGO (mu receptors) or [3H]U69, 593 kappa receptors) in mouse whole-brain assays. The ratios of the calculated Ki values for SNC 80 at mu/delta and kappa/delta sites were 495- and 248-fold, respectively, which indicates a significant degree of delta selectivity for this compound in radioligand binding assays. SNC 80 produced dose- and time-related antinociception in the mouse warm-water tail-flick test after i.c.v., i.th. and i.p. administration. The calculated A50 values (and 95% C.I.) for SNC 80 administered i.c.v., i.th. and i.p. were 104.9 (63.7-172.7) nmol, 69 (51.8-92.1) nmol and 57 (44.5-73.1) mg/kg, respectively. The i.c.v. administration of SNC 80 also produced dose- and time-related antinociception in the hot-plate test, with a calculated A50 value (and 95% C.I.) of 91.9 (60.3-140.0) nmol.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesics↗
PubMed1995

Opioid antinociception in a rat model of visceral pain: systemic versus local drug administration.

Antinociceptive effects of systemically or locally administered opioid mu, kappa and delta agonists were evaluated in a rat model of visceral pain. Resiniferatoxin (RTX, 3 nmol), a capsaicin-like mutant, produced abdominally directed grooming behavior after direct administration into the urinary bladder (intravesical, Lves.) by indwelling cannula. Systemic (s.c. or i.p.) pretreatment with the mu agonists morphine or [D-Ala2, NMePhe4, Gly-ol]enkephalin (Damgo), the kappa agonists trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488) or [5R-(5,7,8-beta)]-N-methyl-N-[7-(1-pyrrolidinyl)1-oxaspiro[4,5]dec - 8-yl]-4-benzofuranacetamide (CI-977), or the nonpeptidic delta agonist (+/-)-4-((alpha-R*)-alpha-((2S*,5R(*)-4-Allyl-2,5-dimethyl-1- piperazinyl)-3-hydroxybenzyl)-N, N-diethylbenzamide (BW373U86) dose-dependently decreased RTX-induced abdominal licking; such antinociception was selectively blocked by the appropriate receptor-selective antagonists beta-funaltrexamine (mu), nor-binaltorphimine (kappa) and naltrindole (delta). Local (i.ves.) BW373U86, [D-Ala2,Glu4]deltorphin (DELT II) and Cl-977 also significantly decreased RTX-induced licking. Intracerabroventricular quaternary naloxone partially blocked the effects of systemic morphine, but not that of CI-977 or BW373U86. Intraperitoneal quaternary naloxone blocked the effect of local and systemic BW373U86 but not that of local or systemic CI-977; systemic morphine was partially blocked. Thus, systemic mu, kappa and delta agonists all produced antinociception against a novel visceral chemical stimulus in the rat. Local CI-977 also produced antinociception, but the only compound clearly acting at peripheral opioid receptors was BW373U86, a delta agonist. This study suggests that opioid delta receptors may be present on bladder nociceptive afferents and may be activated for production of peripheral analgesia.

Analgesics, Opioid↗
PubMed1994

Binding and cAMP studies of melanotropin peptides with the cloned human peripheral melanocortin receptor, hMC1R.

Binding and stimulation of cAMP by the melanotropin peptides alpha-MSH (alpha-melanocyte-stimulating hormone) and its superpotent analogues [Nle4, DPhe7]alpha-MSH (MT-I) and Ac-[Nle4,[formula: see text]alpha-MSH4-10-NH2 (MT-II) were undertaken to examine their respective properties on the human peripheral melanocyte melanocortin receptor, hMC1R. alpha-MSH was found to possess a binding IC50 value of 6.5 +/- 0.9 x 10(-9) M and cAMP EC50 value of 2.0 +/- 0.6 x 10(-9) M. MT-I possesses a binding IC50 value of 1.2 +/- 0.3 x 10(-9) M and a cAMP EC50 of 0.5 +/- 0.03 x 10(-9) M. MT-II possesses a binding IC50 of 0.57 +/- 0.08 x 10(-9) M and cAMP EC50 value of 0.20 +/- 0.05 x 10(-9) M.

Affinity Labels↗
PubMed1994

Design, synthesis, and biological properties of highly potent cyclic dynorphin A analogues. Analogues cyclized between positions 5 and 11.

We have recently reported the synthesis of several cyclic disulfide bridge-containing peptide analogues of dynorphin A (Dyn A), which were conformationally constrained in the putative address segment of the opioid ligand. Several of these analogues, bridged between positions 5 and 11 of Dyn A1-11-NH2, exhibited unexpected selectivities for the kappa and mu receptors of the central over the peripheral nervous systems. In order to further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, we have synthesized a systematic series of Dyn A1-11-NH2 analogues incorporating the sulfydryl containing amino acids L- and D-Cys and L- and D-Pen in positions 5 and 11, thus producing 16 cyclic peptides. In addition, Dyn A1-11-NH2, [D-Leu5]Dyn A1-11-NH2, and [D-Lys11]Dyn A1-11-NH2 were synthesized as standards. Several of these cyclic analogues, especially c[Cys5,D-Cys11] Dyn A1-11-NH2, c[Cys5, L- or D-Pen11]Dyn A1-11-NH2, c[Pen5, L-Pen11]Dyn A1-11-NH2 and c[Pen5, L- or D-Cys11]Dyn A1-11-NH2, retained the same affinity and selectivity (vs the mu and delta receptors) as the parent compound Dyn A1-11-NH2 in the guinea pig brain (GPB). These same analogues and most others exhibited a much lower activity in the guinea pig ileum (GPI), thus leading to centrally vs peripherally selective peptides, but showed a different structure-activity relationship than found previously. In a wider scope, this series of analogues also provided new insights into which amino acids (and their configurations) may be used in positions 5 and 11 of Dyn A analogues for high potency and good selectivity at kappa opioid receptors. The results obtained in the GPB suggest that requirements for binding are not the same for the kappa, mu, or delta central receptors.

Amino Acid Sequence↗
PubMed1994

[N-methylnorleucine-(28,31)]cholecystokinin-(26-33) (SNF 8702) activity at a cloned rat CCKB receptor.

[N-methyl-Nle 28,31)]cholecystokinin-(26-33) (SNF 8702) is a highly selective ligand for the CCKB type of receptor present in the vertebrate central nervous system. Radioligand binding data showing that SNF 8702 binding affinity is reduced by the GTP analog guanylyl-imidodiphosphate suggest that SNF 8702 is an agonist but the ability of SNF 8702 to activate CCKB receptors has not been demonstrated. The present study shows that SNF 8702 is a highly potent agonist at rat CCKB receptors expressed on COS-7 cells and that these receptors are coupled to the mobilization of intracellular calcium. The A50 measured for SNF 8702-induced calcium mobilization (66 pM) is over 6-fold less than that of cholecytstokinin octapeptide (420 pM). Data are also presented showing that SNF 8702 has high binding affinity for these receptors with a Kd value (760 pM) consistent with previous measurements using guinea pig brain tissue preparations.

Animals↗
PubMed1994

Glycopeptide enkephalin analogues produce analgesia in mice: evidence for penetration of the blood-brain barrier.

Most peptides have not proved useful as neuroactive drugs because they are blocked by the blood-brain barrier and do not reach their receptors within the brain. Intraperitoneally administered L-serinyl beta-D-glucoside analogues of [Met5]enkephalin (glycopeptides) have been shown to be transported across the blood-brain barrier to bind with targeted mu- and delta-opioid receptors in the mouse brain. The opioid nature of the binding has been demonstrated with intracerebroventricularly administered naloxone. Paradoxically, glucosylation decreases the lipophilicity of the peptides while promoting transport across the lipophilic endothelial layer. It is suggested that glucose transporter GLUT-1 is responsible for the transport of the peptide message. Profound and long-lasting analgesia has been observed in mice (tail-flick and hot-plate assays) with two of the glycopeptide analogues when administered intraperitoneally.

Amino Acid Sequence↗